Fluorescence microscopy was performed as described (Takaki et al., 2013; Yang et al., 2012 ). Quantification of bacterial burdens, assessments of mycobacterial cording, and enumeration of neutrophils were performed with a Nikon Eclipse Ti-E inverted microscope fitted with 2×, 10×, and 20× objectives. Enumeration of macrophages, assessments of intracellular bacterial growth, measurements of granuloma diameter, and evaluation of histological sections were performed on a Nikon Eclipse E600 upright microscope fitted with 10× and 20× objectives. For laser scanning confocal microscopy, larvae were anesthetized in N-phenylthiourea (PTU)-supplemented fish water containing 0.025% Tricaine and embedded in 1.5% low-melting-point agarose on optical bottom plates or dishes (MatTek Corporation). A Nikon A1 confocal microscope with a 20× Plan Apo 0.75 NA objective was used to generate 40 μm z stacks consisting of 1.3–2 μm optical sections. The galvano scanner was used for all static imaging and for time-lapse imaging of the CHT. Time-lapse images were taken at 5 min intervals for 8 hr. Data were acquired with NIS Elements (Nikon). Macrophage tracks were generated using the 3D tracking feature of Imaris (Bitplane Scientific Software).
Eclipse e600 upright microscope
Manufactured by Nikon
The Eclipse E600 is an upright microscope manufactured by Nikon. It is designed for a variety of laboratory applications, providing high-quality optical performance and versatility. The microscope features an ergonomic design and a range of illumination and imaging options to support various scientific and research needs.
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Lab products found in correlation
Paromomycin, by Merck Group (2 mentions)
Pentr d, by Thermo Fisher Scientific (2 mentions)
Retigaex charge coupled device camera, by PerkinElmer (2 mentions)
Openlab software, by PerkinElmer (2 mentions)
Eclipse ti e inverted microscope, by Nikon (1 mentions)
Optical bottom plates or dishes, by MatTek (1 mentions)
Low melting point agarose, by MatTek (1 mentions)
A1 confocal microscope, by Nikon (1 mentions)
Nis element, by Nikon (1 mentions)
Normal donkey serum (nds), by Jackson ImmunoResearch (1 mentions)
Alexa fluor 488 conjugated donkey anti chicken, by Jackson ImmunoResearch (1 mentions)
Rabbit anti ha, by Cell Signaling Technology (1 mentions)
Rabbit anti iba1, by Fujifilm (1 mentions)
Chicken anti gfp, by Abcam (1 mentions)
Tissue tek oct compound, by Sakura Finetek (1 mentions)
Mounting medium, by Agilent Technologies (1 mentions)
8 protocols using eclipse e600 upright microscope
1
Fluorescence Microscopy for Mycobacterial Infection
2
Immunohistochemistry of Hypothalamic Sections
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Hypothalamic sections were washed with PBS, blocked with 5% normal donkey serum (Jackson Immunoresearch Laboratories), incubated overnight with rabbit anti-Iba1 (1:1,000; Wako Pure Chemicals 019–19741), rabbit anti-HA (1:1,000; Cell Signaling C29F4), chicken anti-GFP (1:5,000; Abcam 13970) and 1 h with appropriate fluorescent secondary antibodies (Alexa Fluor 594-labelled anti-rabbit secondary antibody (1:500; Invitrogen); Alexa Fluor 488-conjugated donkey anti-chicken (1:500; Jackson Immuno Research). Images were captured on an Eclipse E600 upright microscope equipped with a colour digital camera (Nikon).
3
Histological Analysis of MCS
4
Melanin Nanoparticle Uptake in DCs
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Both GM-CSF DCs and FLT3L DCs were incubated for 24 h at 37 °C. This was conducted at 5% CO2 in 12-wells plates at 5 × 105 cells/well, in a volume of 2 mL/well with the indicated solutions. Solutions were used at the following concentrations: 4 μg/mL melanin nanoparticles, 2 μg/mL pOVA30, 1 μg/mL CpG. After incubation, cells were washed, transferred into Superfrost Plus slides (VWR® Superfrost® Plus Micro Slides), and stained with Fontana-Masson by using a stain kit (Sigma Aldrich, Saint Louis, MO, USA) following the manufacturer’s recommendations. Hematoxylin–eosin staining was then performed, and slides were washed and covered with cover slip (Marienfield, Germany) by using a mounting medium (Dako, Santa Clara, CA, USA). Images were obtained using a Nikon’s Eclipse E600 upright microscope with 100× objective. The degree of melanin phagocytosis by cells is presented as the mean percentage of cells internalizing melanin or pOVA30-Mel nanoaggregates in two independent experiments, where the percentage is calculated as the number of cells with melanin in 10 random images (100×) divided by the whole number of cells, for each condition.
5
Inducible YFP-Tagged HPL Expression in Tetrahymena
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The coding regions of HPL genes were amplified from genomic DNA by PCR and inserted into pENTR/D by topoisomerase mediated cloning reactions (Invitrogen/Life Technologies, Carlsbad, CA), then transferred into pICY‐gtw by LR Clonase II recombination as previously described (Malone et al. 2008 ). Oligonucleotides used to amplify each are listed in Table 1 . These YFP expression constructs were introduced into Tetrahymena cells by conjugative electroporation (Gaertig and Kapler 2000 ; Gaertig et al. 1994 ). Transformants were selected and propagated in medium containing 100 μg/ml paromomycin (Sigma‐Aldrich, St. Louis, MO). Expression of Hpl‐YFP proteins was induced by addition of 0.5–1 μg/ml CdCl2 in growing cells or 0.03–0.1 μg/ml in mating cells. To prepare for mating, cells were then washed, starved (11–12 h at 30 °C), and mixed in 10 mM Tris medium with wild‐type strains CU428 or B2086. For epifluorescence microscopy, cells were harvested by low‐speed centrifugation (1,000 g) and immobilized in ~6 μl of 2% methylcellulose. Slides were viewed under a 60X oil immersion lens on a Nikon Eclipse E600 upright microscope. Differential interference contrast (DIC) and YFP fluorescence images were captured using a Qimaging RetigaEX charge‐coupled‐device camera (Burnaby, BC, Canada) and Openlab software (PerkinElmer, San Jose, CA).
6
Visualizing HPL Protein Expression in Tetrahymena
7
Holographic Microscopy for Cell Imaging
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The holographic microscopy setup was similar to that used previously (Singh et al., 2018 (link); Kühn et al., 2018 (link); Thornton et al., 2020 (link)), with a few modifications. The samples were imaged on a Nikon Eclipse E600 upright microscope. The illumination source was a single-mode fibre-coupled laser diode with peak emission at 642 nm. The end of the fibre was mounted below the specimen stage using a custom adaptor and delivered a total of 15 mW of optical power to the sample. A Mikrotron MC-1362 monochrome camera was used to acquire videos of 3000 frames, at a frame rate of 50 Hz and with an exposure time of 100 μs. A 10× magnification bright-field lens with numerical aperture of 0.3 was used to acquire data at a video resolution of 1024 × 1024 pixels2, corresponding to a field of view measuring 1.44 × 1.44 mm2. The raw videos were saved as uncompressed, 8-bit AVI files. Three biological replicates of each condition were prepared, with five technical replicates from each of these.
8
Histological Analysis of Bone Defects
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